Na,K-ATPase is a ubiquitous transmembrane protein that regulates and maintains the intracellular Na(+) and K(+) gradient necessary for cell homeostasis. Recently, the importance of this pump in external stimuli-induced leukemia cell apoptosis has been increasingly appreciated, however, the exact role of Na,K-ATPase in mitochondrial apoptotic pathway still remains little understood. In this study, we found mitochondrial toxin rotenone caused a rapid mitochondrial membrane potential (MMP) collapse in Jurkat cells followed by plasma membrane depolarization (PMP). Similar results were also obtained in human U937 cells and non-cancerous mouse primary T cells. Rotenone-induced PMP depolarization occurred before apoptosis and well correlated with Na,K-ATPase impairment. To understand the mechanisms, Jurkat cells with mtDNA depletion and catalase overexpression were used. The results demonstrated that both PMP depolarization and Na,K-ATPase impairment induced by rotenone were regulated by mitochondrial H(2)O(2) and Bcl-2. Finally, Na,K-ATPase suppression by ouabain greatly accelerated and enhanced mitochondrial toxins-induced cells apoptosis in Jurkat, U937 and primary T cells. In sum, by using leukemia cells and mouse primary T cells, we confirmed that mitochondria-to-Na,K-ATPase and PMP depolarization might represent a novel mechanism for mitochondria to amplify death signals in the initiation stage of cells apoptosis induced by mitochondrial toxins.